United States Patent [191
`Bonaventura et al.
`
`[54] PROCESSES FOR EXTRACTING OXYGEN
`FROM FLUIDS USING IMMOBILIZED
`HEMOGLOBIN
`
`[75]
`
`Inventors: Joseph Bonaventura; Celia
`Bonaventura, both of Beaufort, N2C.
`[73] Assignee: Duke University, Durham, N.C.
`[21] Appl. No.: 372,338
`Apr. 27, 1982
`
`[22] Filed:
`
`Related U.S. Application Data
`
`[62] Division of Set. No. 196,036, Oct. 10, 1980, Pat. No.
`4,343,715.
`
`Int. C1.3 .............................................. C01B 13/00
`[51]
`[52] U.S. CI ...................................... 23/293 R; 55/68;
`210/321.4
`[58] Field of Search ........................ 424/177; 423/579;
`55/68; 210/321.4; 422/48, 122; 23/293 R
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,925,344 12/1975 Mazur ................................. 424/177
`4,053,590 10/1977 Bonsen et al ....................... 424/177
`4,061,736 12/1977 Morris et al ........................ 424/177
`
`[11]
`[45]
`
`4,427,416
`Jan. 24, 1984
`
`4,064,1!8 12/1977 Wong .................................. 424/177
`
`OTHER PUBLICATIONS
`
`Lampe et al., "Der Einflub der Immobilisierung Von
`H~imoglobin auf dessen Sauerstoffbindung," Acta Biol.
`Med. Germ., Band 34, Seite 359-363 (1975).
`Lampe et al., "Untersuchungen zur Bindung Von
`Sauerstoff an TrSgerfixiertes H~imoglobin," Studia Bio-
`physica, Berlin, Band 39, 1973, Heft 1, S. 19-24, Printed
`in the German Democratic Republic.
`Antonini et al., "Immobilized Hemoproteins," Immobi-
`lized Enzymes, vol. XLIV (1976), pp. 538-546, Aca-
`demic Press.
`
`Primary Examiner--Edward J. Meros
`Assistant Examiner--Wayne A. Langel
`Attorney, Agent, or Firm--Oblon, Fisher, Spivak,
`McClelland & Maier
`
`[57]
`
`ABSTRACT
`
`An oxygen carrier, capable of reversibly binding and
`releasing oxygen, immobilized in a polymer matrix and
`a method of recovering dissolved oxygen from fluids
`utilizing the same.
`
`21 Claims, 13 Drawing Figures
`
`OXYGEN ELECTRODE
`IN FLOW CELL
`
`~ OXYGEN METER
`
`IMMOBILIZED
`HEMOGLOBIN
`
`STRIPCNARTRECOROERMANIFOLD
`
`/
`
`AIR EQUILIBRATED SEA WATER
`
`DE-AERATED SEA WATER
`
`DITHIONITE IN SEA WATER
`
`FERRICYANIDE IN SEA WATER ~
`
`Akermin, Inc.
`Exhibit 1005
`Page 1
`
`

`
`U.S. Patent Jan. 24, 1984
`
`Sheet ~ of 8
`
`4,427,416
`
`FIG.
`
`Akermin, Inc.
`Exhibit 1005
`Page 2
`
`

`
`U.S. Patent Jan. 24, 1984
`
`Sheet 2 of 8
`
`4,427,416
`
`OXYGEN DISSOLVED
`IN SEAWATER
`
`OXYGEN
`CARRIER
`
`LOADING
`CYCLE
`
`OXYGEN
`CARRIER
`
`REGENERATION
`CYCLE
`
`UNLOADING
`CYCLE
`
`OXYGEN FOR
`HUMAN
`CONSUMPTION
`
`Akermin, Inc.
`Exhibit 1005
`Page 3
`
`

`
`U.S. Patent Jan. 24, 1984
`
`Sheet 3 of 8
`
`4,427,416
`
`OXYGEN ELECTRODE
`IN FLOW CELL
`
`OXYGEN METER
`
` ool
`
`STRIP CHART RECORDER
`
`IMMOBILIZED
`HEMOGLOBIN
`REACTOR
`
`PUMP
`
`/ ~
`
`MANIFOLD
`
`AIR EQUILIBRATED SEA WATER L.~
`
`II
`
`DE-AERATED SEA WATER
`
`DITHIONITE IN SEA WATER
`
`FERRICYANIDE IN SEA WATER
`
`Akermin, Inc.
`Exhibit 1005
`Page 4
`
`

`
`U.S. Patent jan. 24, 1984
`
`Sheet 4 or 8
`
`4,427,4 16
`
`12-
`
`10
`
`8
`
`6
`
`4
`
`2
`
`0
`
`~00
`
`I I
`200 300 400 500 600
`VOLUME OF SEAWATER (rnl) THROUGH
`COLUMN IN LOADING CYCLE
`
`I
`700
`
`I
`80O
`
`MATRIX: SIZED- GEL FORMULATION OF HEMOSPONGE
`COLUMN: 75rnl~ 2.26g HEMOGLOBIN
`SEAWATER FLOW RATE: 26.5ml/rnin.
`ABSORBED OXYGEN: 2.66rag 162% OF THEORETICAL MAXIMUM)
`
`FI~.6
`
`WHERE ,~ " ~/ = OXYGEN PASSING COUJMN A AND
`ABSORBED BY BA CKUP COLUMN B
`
`lO0
`
`50
`
`~URVE
`FOR COLUMN
`
`I I I
`12
`8
`t6
`TIME (MINUTES)
`
`I
`20
`
`FIG. 8
`
`Akermin, Inc.
`Exhibit 1005
`Page 5
`
`

`
`U.S. Patent Jan. 24, 1984
`
`Sheet 5 of 8
`
`4,427,416
`
`UNLOADED
`
`~5
`
`5
`
`I I I I
`100 200 300 400
`
`VOLUME OF SEAWATER (rnl) THROUGH
`COLUMN 1N UNLOADING CYCLE
`
`MATRIX: SIZED-GEL FORMULATION OF I-IEMOSPONGE
`COLUMN: 75ml, 2.26g HEMOGLOBIN
`SEAWATER FLOW RATE: 26.Sml/min. WITH 0.25g
`FERRICYANIDE IN SEA WATER
`RELEASED OXYGEN: 1.98rng (46% OF THEORETICAL
`MAXIMUM)
`
`FIG. 7
`
`Akermin, Inc.
`Exhibit 1005
`Page 6
`
`

`
`U.$o Patent Jan. 24, 1984
`
`Sheet 6 of 8
`
`4,427,416
`
`OUTLET
`
`OUTLET
`
`FIG. 9
`
`INLET FOR
`CHEMICAL ADDITIVE
`
`OUTLET ~
`
`COMPRESSED HEMOSPONGE
`
`FLUID
`INLET
`
`BELT OF
`HEMOSPONGE
`
`;HEMICAL COMPARTMENT
`FOR RECYCLING
`
`FIG. It
`
`Akermin, Inc.
`Exhibit 1005
`Page 7
`
`

`
`UoS. Patent Jan. 24, 1984
`
`Sheet 7of8
`
`4,427,416
`
`0.75
`
`0.50
`
`0.25
`
`0
`
`L06 P02
`
`Akermin, Inc.
`Exhibit 1005
`Page 8
`
`

`
`U.S. Patent Jan. 24, 1984
`
`Sheet 8 of 8
`
`4,427,416
`
`0.5
`
`I
`
`-0.5
`
`I
`
`0.0
`
`I I
`
`0.5
`
`1.0
`
`FIG.
`
`Akermin, Inc.
`Exhibit 1005
`Page 9
`
`

`
`4,427,416
`
`PROCESSES FOR EXTRACTING OXYGEN FROM
`FLUIDS USING IMMOBILIZED HEMOGLOBIN
`
`This is a division, of application Ser. No. 196,036,
`filed Oct. 10, 1980 now U.S. Pat. No. 4,343,715.
`
`BACKGROUND OF THEINVENTION
`
`1. Field of the Invention
`This invention relates to a material for and a process
`of extracting oxygen from fluids, e.g., gases and natural
`waters, such as, in which the oxygen is dissolved.
`2. Description of the Prior Art
`One of the primary problems which hinders man in
`his efforts to explore and develop the ocean realms is
`the lack of a ready supply of oxygen. In most of the
`world’s oceans, the oxygen content of both shallow and
`deep waters is similar to that of surface water in equilib-
`rium with air. Practical methods have not yet been
`devised for extracting and utilizing this vast amount of
`oxygen for the maintenance of man in an undersea envi-
`ronment. Fish, however, have obviously solved thb
`problem of oxygen extraction from seawater. Fish spe-
`cies weighing well over a thousand pounds and burning
`metabolities at rates roughly comparable to that of man
`easily extract adequate dissolved oxygen from seawater
`for their varied activities. Moreover, many species of
`fish transfer oxygen from seawater into a gaseous state.
`These fish, ones that possess swim bladders, are able to
`pump and concentrate oxygen against enormous hydro-
`static pressure gradients. In certain fish species oxygen
`is transported from the dissolved state in seawater, with
`a pO2of 0.2 atmospheres, to a gaseous phase in the swim
`bladder where the pO2 may exceed 100 atmospheres.
`The transfer of oxygen from the seawater to the swim
`bladder is made possible by the presence of specialized
`hemoglobin molecules in fish erythrocytes. These spe-
`cialized hemoglobin molecules-called Root effect
`hemoglobins-act as miniature molecular pumps. The
`driving force for such a pump is metabolically produced
`lactic acid and various organic phosphate cofactors.
`However, we cannot directly mimic these biological
`systems, since the hemoglobin is circulated in the blood
`and is consequently not in a form which can be easily
`manipulated in large scale flow systems. Many attempts
`to develop methodologies of extracting oxygen from
`gaseous mixtures or water are known. Warne e~ al, U.S.
`Pat. No. 2,217,850, and Fogler et al, U.S. Pat. No.
`2,450,276, disclose processes of separating oxygen from
`other gases using solutions of cobalt compounds. How-
`ever, these techniques would be ineffective in a liquid
`system, e.g., seawater, since the compounds are in solu-
`tion and would be washed away. Miller, U.S. Pat. No.
`3,230,045, discloses using oxygen-binding chromopro-
`reins such as hemoglobin and hemocyanin to separate
`oxygen from other gases. The chromoproteins are kept
`moist or in solution and are immobilized on filter paper
`where they may be bound by a binder such as fibrin, and
`an electrolyte such as sodium chloride may be present.
`However, this technique would also be ineffective in a
`liquid system since the protein is not insoluble and thus
`would be washed away if water was allowed to flow
`through the system. Moreover, there is no provision for
`regeneration of oxidized (inactive) oxygen carriers.
`Bodell, U.S. Pat. No. 3,333,583, and Robb, U.S. Pat.
`No. 3,369,343, disclose apparatus for extracting oxygen
`from seawater using thin tubes of silicone rubber or
`membrane of silicone rubber, respectively. However,
`
`2
`neither the capillary networks nor the permeable mem-
`branes have been found to be practicable in real-life
`situations. Isomura, U.S. Pat. No. 3,377,777, discloses
`concentrating oxygen from natural waters by equilibra-
`5 tion with exhaled gases, i.e. by utilizing large areas of
`gas-water interface and simple diffusional consider-
`ations such that the partial pressure of the gas phase and
`the partial pressure of the liquid phase in the extraction
`zone provide for release of oxygen from the liquid
`10 phase into the gas phase and absorption of CO2 by the
`water phase. Additionally, the solubility of oxygen in
`seawater is decreased by heating the seawater and this
`heating also increases the solubility of CO2. However,
`the heating of the seawater produces an energetically
`i5 undesirable process. Rind, U.S. Pat. No. 4,020,833, dis-
`closes an oxygen source for closed environments com-
`prising a mixture of a metallic superoxide, which re-
`leases oxygen upon contact with CO2 and water vapor,
`and a material which absorbs CO2. However, this sys-
`20 tem suffers from the defect of the capacity being limited
`by the bulk amount of mixture which can be carried.
`Iles et al, U.S. Pat. No. 4,165,972, discloses separating
`oxygen from gas mixtures using metal chelates as sor-
`bents. However, the technique is not extendable to the
`25 extraction of oxygen from water.
`Many compounds in solution have been examined
`with respect to their oxygen absorption properties and
`the mechanistics thereof. The properties of hemoglo-
`bins, hemerythrins and hemocyanins, the naturally oc-
`30 curring oxygen carriers, have been the subject of nu-
`merous studies, as documented in Bonaventura et al, J.
`Am. Zool., 20, 7 [1980] and 20, 131 (1980). Artificial
`oxygen carriers and their properties in solution are
`described by a number of researchers. Traylor et al,
`35 "Solvent Effects on Reversible Formation and Oxida-
`tive Stability of Heme-Oxygen Complexes", J.A.C.S.,
`96, 5597 (1974) discloses the effect of solvent polarity
`on oxygenation of several heme-base complexes pre-
`pared by reduction with sodium dithionite or a mixture
`40 of Pd black and calcium hydride. Crumbliss et al, "Mo-
`nomeric Cobalt-Oxygen Complexes", Science, 6, June
`1969, Volume 164, pp. 1168-1170, discloses Schiff base
`complexes of Co(II) which form stable cobalt-oxygen
`species in solution instead of cobalt-oxygen-cobalt
`45 bridged complexes. Crumbliss et al, "Monomeric Oxy-
`gen Adducts of N,N’-Ethylenebis
`(acetylacetoniminato) ligand-cobalt(III). Preparation
`and Properties" J.A.C.S. 92, 55 (1970), discloses a series
`of monomeric molecular oxygen carriers based on co-
`50 bait ligand complexes. Dufour et al, "Reaction of In-
`doles with Molecular Oxygen Catalyzed by Metallo-
`porphyrins", Journal of Molecular Catalysis (In Press),
`discloses the catalysis of the oxygenation of simple,
`alkyl-substituted indoles by Co(II), Co(III), and Mn(III)
`55 meso-tetraphenyl-porphins wherein a ternary complex
`O2-CoTPP-indole is formed initially. Brault et al, "Fer-
`rous Porphyrins in Organic Solvents. I. Preparation and
`Coordinating Properties", Biochemistry, 13, 4591
`(1974), discloses the preparation and properties of fer-
`60 rous deutereporphyrin dimethyl ester and ferrous meso:
`tetraphenylporphin in various organic solvents. Chang
`et al, "Kinetics of Reversible Oxygenation of Pyr-
`roheme-N-[3-(1-imidazolyl)propyl] amide", discloses
`studies on the oxygenation of pyrroheme-N-[3-(1-
`65 imidazolyl)propyl] amide, i.e. a synthesized section of
`the myoglobin active site. Castro, "Hexa and Pen-
`tacoordinate Iron Poryhyrins", 13ioinorganic Chemis-
`try, 4, 45-65 (1974), discloses the direct synthesis of
`
`Akermin, Inc.
`Exhibit 1005
`Page 10
`
`

`
`4,427,416
`
`3
`hexa and pentacoordinate iron porphyrins, i.e. the pros-
`thetic groups for the active sites of certain cytochrome
`and globin heine proteins. Chang et al, "Solution Be-
`havior of a Synthetic Myoglobin Active Site", J.A.C.S.,
`95, 5810 (1973), discloses studies on a synthesized sec-
`tion of the myoglobin active site and indicates that the
`oxygen binding reaction does not require the protein.
`Naturally occurring oxygen carriers have been chemi-
`cally cross-linked and their properties described.
`Bonsen et al, U.S. Pat. No. 4,053,590, discloses a poly-
`merized, cross-linked, stromal-free, hemoglobin pro-
`posed to be useful as a blood substitute. Morris et al,
`U.S. Pat. No. 4,061,736, discloses intramolecularly
`cross-linked, stromal-free hemoglobin. Wong, U.S. Pat.
`No. 4,064,118, discloses a blood substitute or extender
`prepared by coupling hemoglobin with a polysaccha-
`ride material. Mazur, U.S. Pat. No. 3,925,344, discloses
`a plasma protein substitute, i.e. an intramolecular, cross-
`linked hemoglobin composition. However cross-linked
`hemoglobin produces macromolecular complexes that
`retain many of hemoglobin’s native properties. The
`cross-linking of hemoglobin results in a product that is
`a solution or a dispersion, is not manipulable or, in fact,
`insolubilized. Large scale flow-thru systems where vol-
`umes of water must flow by or through an oxygen ex-
`tracting medium cannot use hemoglobin which has been
`crosslinked because the hemoglobin is not truly insolu-
`ble. In other words, crosslinking does not accomplish a
`useful insolubilization, in that, even after crosslinking,
`the protein in its final form has the characteristics of a
`fluid.
`Numerous papers have been published on immobili-
`zation of hemoglobin and its functional consequences,
`but not in connection with processes for efficient oxy-
`gen extraction from fluids. Vejux et al, "Photoacoustic
`Spectrometry of Macroporous Hemoglobin Particles",
`J. Opt. Soc. Am., 70, 560-562 (1980), discloses glutaral-
`dehyde cross-linked hemoglobin and its functional
`properties. The preparation is described as being made
`up of macroporous particles. Hallaway et al, "Changes
`in Conformation and Function of Hemoglobin and
`Myoglobin Induced by Adsorption to Silica", BBRC,
`86, 689-696 (1979), discloses that hemoglobin adsorbed
`on silica is somewhat different from hemoglobin in
`solution. The adsorbed form is not suitable for 02 ex-
`traction from liquids. Antonini et al, "Immobilized He-
`moproteins", Methods of Enzymology, 44, 538-546
`(1976), discloses standard immobilization techniques as
`applied to hemoglobin and their functional conse-
`quences. Mention is made of hemoproteins bound to
`cross-linked insoluble polysaccharides such as Sepha-
`dex or Sepharose, using a pre-activation of the resin
`with CNBr. Rossi-Fanelli et al, "Properties of Human
`Hemoglobin Immobilized on Sepharose 4B", Eur. J.
`Biochemistry, 92, 253-259 (1978), discloses that the
`ability of the hemoglobin to be bound to Sepharose 4B
`is dependent upon the conformational state of the pro-
`tein.. Colosimo et al, "The Ethylisocyanate (EIC) Equi-
`librium of Matrix-Bound Hemoglobin", BBA, 328,
`74-80 (1973), discloses Sephadex G-100, Sephadex
`DEAE-A50 and Sephadex CM-CS0 as supports for
`human hemoglobin insolubilization. The paper shows
`that the affinity of the insolubilized protein for EIC is
`increased relative to that in solution. Lampe et al, "Die
`Bindung yon Sauerstoff an tragerfixiertes Hamo-
`globin", Acta Biol. Med. Germ., 33, K49-K54 (1974),
`discloses studies on CM-Sephadex insolubilized hemo-
`globins. Lampe et al, "Der EinfluB der Immobilisierung
`
`von Hamoglobin auf dessen Sauerstoffindung", Acta
`Biol. Med. Germ., 34, 359-363 (1975), discloses studies
`on CM-Sephadex insolubilized hemoglobins. Pommer-
`ening et al, "Studies on the Characterization of Matrix-
`5 Bound Solubilized Human Hemoglobin", Interna-
`tionales Symposium uber Struktur und Funktion der
`Erythrezyten (Rapoport and Jung, ed.), Berlin Akade-
`mie-Verlag Press, 179-186 (1975), discloses Sepharose-
`Sephadex types of insolubilization. Brunori et al, "Prop-
`10 erties of Trout Hemoglobin Covalently Bound to a
`Solid Matrix", BBA, 494(2), 426-432, discloses Se,
`pharose 4B or Sephadex G-200, activated by CNBr, to
`immobilize the hemoglobin. Some.changes in the func-
`tional properties of the hemoglobin were found.
`15 As may be discerned, there are generally two classes
`of "insolubilized" hemoglobins described in patents or
`in open literature. First, cross-linked hemoglobin, e.g.,
`as by glutaraldehyde. Biodegradation of such forms of
`insolubilized hemoglobin would be rapidly accom-
`20 plished by the microorganisms in seawater. Nor has full
`functionality been demonstrated in published accounts.
`This does not mean that functional properties are neces-
`sarily eliminated, but, that methods as described are not
`suitable for achieving an immobilized form with unim-
`25 paired function. Second, Sephadex or Sepharose bound
`hemoglobins. Low hemoglobin content per volume
`(specific capacity) makes these methods of insolubiliza-
`tion untenable for large scale use. Biodegradation prob-
`lems are also present. Additionally,. it is not generally
`30 possible to achieve high flow rates through such materi-
`als.
`Various techniques for the insolubilization (or immo-
`bilization) of biological materials have been developed,
`though not described in conjunction with insolubiliza-
`35 tion and utilization of oxygen carriers. Stanley, U.S.
`Pat. No. 3,672,955, discloses a technique for the prepa-
`ration of an insoluble, active enzyme, a biological cata-
`lyst, wherein an aqueous dispersion of the enzyme is
`emulsified with an organic polyisocyanate, mixed with
`40 a solid carrier and the volatile components are then
`evaporated from the mixture. Wood et al, U.S. Pat. No.
`3,928,138, discloses a method of preparing a bound
`enzyme wherein, prior to foaming, an isocyanate-
`capped polyurethane is contacted with an aqueous dis-
`45 persion of enzyme under foam-forming conditions,
`whereby polyurethane foams containing integrally
`bound enzyme are obtained. Unsworth et al, U.S. Pat.
`No. 3,928,230, discloses the encapsulation of fluids and
`solids by dissolving a water-insoluble polymerizable
`50 epoxy monomer in a solvent having high affinity for
`water; dispersing the monomer solution in water; dis-
`persing in the so-formed aqueous dispersion the sub-
`stance to be encapsulated; adding a polymerizing agent
`in a solvent having a higher affinity for water than for
`55 the polymerizing agent; and polymerizing until poly-
`merization of the monomer is complete. Wood et al,
`U.S. Pat. No. 3,929,574, discloses an enzyme integrally
`bound to a foamed polyurethane parepared by, prior to
`foaming, contacting an isocyanate-capped polyurethane
`60 with an aqueous dispersion of enzyme under foam-
`forming conditions, whereby polyurethane foams con-
`taining integrally bound enzyme are obtained. Hartde-
`gen et al, U.S. Pat. No. 4,094,744, discloses water-dis-
`persible protein/polyurethane reaction products
`65 formed by admixing a water-dispersible, biologically-
`active protein and an isocyante-capped liquid polyure-
`thane prepolymer having a linear polyester backbone
`under essentially anhydrous conditions to form a solu-
`
`Akermin, Inc.
`Exhibit 1005
`Page 11
`
`

`
`4,427,416
`
`6
`FIG. 8 is a representative absorption curve for a two
`column system.
`FIG. 9 is a schematic diagram of an absorption sys-
`tem utilizing a static oxygen carrier, denoted as Hemos-
`ponge.
`FIG. 10 is a schematic diagram of an absorption sys-
`tem utilizing a piston system capable of driving the fluid
`flow from the various inlets and compressing the static
`oxygen carrier, denoted as Hemosponge.
`FIG. 11 is a schematic diagram of an absorption sys-
`tem utilizing a belt system wherein the oxygen carrier,
`denoted as Hemosponge, is not static but is transported
`from regions of high oxygen concentration to regions of
`low concentration or to a region where oxygen release
`is initiated by a chemical means.
`FIG. 12 depicts reversible oxygen binding by human
`hemoglobin in polyurethane gel particles of sizes com-
`parable to those of red blood cells. Y represents fraction
`saturation of the oxygen carrier.
`FIG. 13 shows a spectrophotometric demonstration
`of reversible oxygen binding by a film of hemoglobin
`insolubilized in a polyu__rethane gel. Oxygen extraction
`in this case is from air. Y represents fractional saturation
`of the oxygen carrier with oxygen.
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`
`tion, said protein and prepolymer reacting to form a
`water-soluble reaction product wherein the protein and
`prepolymer are bound together. Hartdegeu et al, U.S.
`Pat. No. 4,098,645, discloses enzymes immobilized by
`the process of mixing the protein and an isocyanate- 5
`capped liquid polyurethane prepolymer in the absence
`of water; foaming the mixture by reacting it with water
`to form a polyurethane foam. Huper et al, U.S. Pat. No.
`4,044,196, discloses proteins insolubilized using poly-
`mers containing maleic anhydride or di- and poly- 10
`methacrylates. Huper et al, U.S. Pat. No. 3,871,964,
`discloses proteins insolubilized using polymers contain-
`ing anhydride, di-methacrylate and a hydrophilic mon-
`omer. However, there is no disclosure in the art of an
`effective way to insolubilize hemoglobin or other oxy- 15
`gen carriers at high concentrations so as to render them
`active, insoluble and manipulable.
`A need therefor continues to exist for not only im-
`proved methods for insolubilizing hemoglobin or other
`oxygen carrying compounds but also for a method of 20
`extracting the available dissolved oxygen from natural
`waters and other fluids. Such methods as will be de-
`scribed will also be useful for preparing blood substi-
`tutes which are capable of reversible oxygen binding 25
`under physiological conditions.
`
`SUMMARY OF THE INVENTION
`
`Accordingly, one object of the invention is to pro-
`vide an insolubilized oxygen carrier which is effective
`for the extraction of oxygen from fluids, e.g., gases and
`natural waters, such as seawater.
`A further object of the invention is to provide an
`oxygen carrier in a form such that oxygen can be car-
`ded from regions of high concentration, such as the
`lungs, and unloaded in regions of low concentration,
`such as the respiring tissues.
`Briefly, these objects and other objects of the inven-
`tion as hereinafter will become more readily apparent
`can be attained by providing oxygen carders which
`have been insolubilized at high concentration by being
`entrapped and/or covalently linked to a polyurethane
`matrix or to comparable suppports in states that are
`capable of reversible oxygen binding and are regenera-
`ble in the event of oxidation.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`A more complete appreciation of the invention and
`many of the attendant advantages thereof will be
`readily obtained as the same becomes better understood
`by reference to the following detailed description when
`considered in connection with the accompanying draw-
`ings, wherein:
`FIG. 1 is a 40X magnification showing the nature of
`a polyurethane reticulated matrix in which hemoglobin
`is incorporated.
`FIG. 2 is a 2400X magnification of the thin walls of
`the matrix of FIG. 1 at a region of contact between two
`of the oval compartments.
`FIG. 3 is a 4000X magnification showing the rippled
`surface of the thin walls of the matrix of FIG. 1. The
`hemoglobin material is held within the walls.
`FIG. 4 is a schematic diagram of an oxygen extrac-
`tion process.
`FIG. 5 is a schematic diagram of a laboratory oxygen
`recovery apparatus.
`FIG. 6 is a representative oxygen loading curve.
`FIG. 7 is a representative oxygen unloading curve.
`
`This invention relates to the incorporation of an oxy-
`gen carrier, which can be a biological macromolecule,
`30 into an insolubilized form, which can be a polymeric
`matrix. More particularly, the preferred embodiment of
`the invention involves a biochemical engineering tech-
`nique known as molecular entrapment. The oxygen
`carrier used by man and other mammals, as well as by
`35 most other vertebrates, is hemoglobin. By molecular
`entrapment, hemoglobin can be made insoluble and
`consequently more amenable for use in a recycling and
`regenerable system. Optimally, entrapment is analogous
`to placing a cage around the biologically active mate-
`40 rial. This cage, or network, entraps the material but
`does not render it inactive. The entrapment insolubilizes
`the material and renders it amenable to manipulation.
`The degree to which function is maintained varies
`greatly with the type of entrapment process used. In the
`45 preferred polyurethane matrices of this invention, the
`material retains essentially full biological activity. The
`preferred material for hemoglobin insolubilization is a
`hydrophilic polyurethane. The word polyurethane is
`all-inclusive and is used for all polymers containing
`50 urethane linkages. Most polyurethane foams do not
`have as their starting point a water soluble component,
`and consequently are not compatible with most biologi-
`cal materials. A special kind of polyurethane prepoly-
`met, one which polymedzes when in contact with wa-
`S5 ter, is required to create the material designated as the
`preferred embodiment of this invention. In FIGS. 1-3
`and 6-11, the insolubilized oxygen carrier is referred to
`as Hemosponge. Polymerization and insolubilization of
`the oxygen carrier is effected by adding water to the
`60 pre-polymer. The hydrophilic urethane prepolymer
`which was used to create the Hemosponge described
`here was developed by W. D. Grace and Company and
`is the commercially available material HYPOL. Other
`urethane monomers compatible with the water soluble
`65 nature of hemoglobin can be synthesized and these
`other materials may be used in creation of Hemosponge.
`The means by which such hydrophilic foams can be
`synthesized have been published and are in the public
`
`Akermin, Inc.
`Exhibit 1005
`Page 12
`
`

`
`4,427,416
`
`domain. The advantages of this technique are numer-
`ous. Hemoglobin in such polyurethane foams or gels
`can be entrapped in high concentrations, the matrix
`having a variable degree of reticulation. The matrix in
`the formulations described herein is durable and amena-
`ble to all sorts of mechanical manipulation. It can be
`formed into virtually any configuration. It can be cut,
`machined, drilled, etc. Even more importantly, in the
`form of a sponge or sized-gel particles, it has very good
`diffusional and fluid-flow characteristics.
`During the insolubilization process, using the pre-
`ferred polyurethane matrix, the amount of CO2 libera-
`tion can be varied over a very wide range. When CO2
`liberation is abundant, the resultant material is highly
`reticulated and spongy as illustrated in FIGS. 1-3. High
`flow rates are typical with such reticulated forms.
`When formulated with a minimum of CO2 liberation,
`polyurethane gels can be formed without reticulation.
`For a typical insolubilization of hemoglobin, the proce-
`dure would be:
`(1) Pack red blood cells by centrifugation.
`(2) Wash cells twice with physiological saline and
`repack.
`(3) Lyse with distilled water to a concentration of
`hemoglobin of about 125 mg/ml and adjust the pH
`to 6.0 with dilute HC1.
`(4) Remove red cell membranes by centrifugation to
`save the hemoglobin solution.
`(5) Mix 20 ml of hemoglobin solution with 4 ml
`HYPOL.
`(6) Allow to form a non-reticulated gel at room tem-
`perature (20*-25* C.).
`The resultant gel material can be ground and sorted into
`gel particles of defined size. Such gel particles of ap-
`proximately 0.5 mm diameter have been found to have
`good diffusional and flow-rate characteristics. Good
`oxygen loading curves as shown in FIGS: 6-7 have
`been obtained using packed gel particle columns at flow
`rates of about one column volume per minute in devices
`illustrated in FIG. 5. The diffusional characteristics of
`such columns have been found to be dependent upon
`gel size, and such gel size is readily optimizable. Addi-
`tionally, the use of sized-gel particles allow the use of
`fluidized bed absorption, which greatly increases the
`flow. rate of oxygen-containing water. Furthermore,
`hemoglobin can be loaded onto the sized-gel particles at
`high concentrations. Up to 30 g hemoglobin/1000 ml of
`column volume can be attained without problems with
`flow rates or diffusional difficulties. Overall yield of
`oxygen from such columns gives about 50-70% of the
`theoretical maximum based on the amount of insolubil-
`ized hemoglobin which the column contains. The pH
`during gel formation is quite important. The optimum
`pH for the insolubilization process appear to be about
`pH 6.
`Hemoglobin and other oxygen carriers may be insolu-
`bilized at high concentration and with reversible oxy-
`gen binding characteristics in other than polyurethane
`matrices. Acrylic gels may also be used in this inven-
`tion, especially the hydrophilic acrylates. Also reactive
`polymers containing maleic anhydride as one of the
`constituents are highly suitable for covalently binding
`hemoglobin or alternate oxygen carriers. Such poly-
`mers give high bonding yields (1 gr/gr polymer) and
`the resulting material exhibits reversible oxygen bin-
`ding/unloading. These polymers are particularly suit-
`able in the form of macroporous beads. Two other reac-
`tive polymers which may be used are the epoxy type,
`
`made by polymerization of glycidyl methacrylate as a
`copolymer, and the glutaronic aldehyde type, from
`pyridine-containing polymers reacted with cyanogen
`bromide. Additionally, hemoglobin and other oxygen
`5 carriers can be covalently bound to various insoluble
`matrices by other techniques, such as those used for
`enzyme immobilization, such as are described in Meth-
`ods in Enzymology, Volume 44, Immobilized Enzymes,
`Academic Press, New York (1976). In these alternative
`10 methods of insolubilization, successful formulations
`must have the characteristics of durability, resistance to
`biodegradation, high specific density of hemoglobin on
`the carrier, high flow rates with little pressure drop, and
`good diffusional characteristics. Thus, Sephadex or
`15 Sepharose bound hemoglobin forms previously de-
`scribed are inferior methods for hemoglobin insolubili-
`zation.
`Hemoglobin is, of course, by far the most common
`oxygen carrying protein found in nature. Within this
`20 context, however, it is possible to use in commercial
`applications any of the hemoglobins which are available
`in large quantity, e.g., human, bovine, porcine and
`equine hemoglobins. Further, whole blood, lysed cells,
`stripped or unstripped hemolysates can be used. Modi-
`25 fied forms of hemoglobin, i.e. high or low affinity hemo-
`globins, as known in the art, are also useful. Hemoglo-
`bin can be treated to manipulate its affinity. Covalent or
`chemical modification, prior to immobilization, or treat-
`ment or the immobilized hemoglobin with cofactors
`30 that bind tightly and alter oxygen binding affinity (these
`are removable by washing the polymeric matrix with
`appropriate buffers) can be used. Additives, like cata-
`lase, superoxide dismutase and methemoglobin reduc-
`tase, can be added to the solutions of hemoglobin prior
`35 to insolubilization in the polymeric matrix. These agents
`are normally found in red blood cells and can be useful
`in conferring structural and functional stability to the
`insolubilized hemoglobin. Additionally, reagents such
`as glycerol, which are known to impart structural stabil-
`40 ity to proteins in solution, can be usefully added to the
`solution of hemoglobin prior to incorporation into the
`polymeric matrix or, likewise, prior to covalent attach-
`ment to other polymeric supports.
`Although hemoglobin is by far the most common
`45 oxygen carrier found in nature, other types of oxygen
`carriers are found in a number of species. In particular
`hemocyanin and hemerythrins are known and useable
`although they suffer from the deficiency of being un-
`vailable in large quantities.
`50 The use of synthetic oxygen carriers, such as the
`modified hemes described earlier and other like com-
`pounds known in the art, which show reversible oxygen
`binding, allow the attainment of high oxygen absorbing
`capacity in minimum absorber volume. These com-
`55 pounds are particularly useful when eovalently bound
`to a polymeric matrix.
`While mere contact with dissolved oxygen is suffi-
`cient for oxygen loading of the oxygen carriers of this
`invention, many variations are possible in the unloading
`60 cycle. A chemical alteration which oxidizes or inacti-
`vates the oxygen carrier is able to cause release of all of
`the bound oxygen. For example, f